1. Technical Field
This invention relates to plant extracts having therapeutic and other uses, and more specifically to an extract of juice from berries of the Vaccinium plant genus having an anti-microbial-adhesion activity.
2. Background Art
Adhesion of the bacteria to each other (intraspecies) and to other bacterial species (intergeneric coaggregation) as well as to host tissues and cells contributes significantly to disease progression and pathology as for example in dental caries and plaque as well as in the persistance of Helicobacter pylori infection. It would therefore be useful to have compounds which can interrupt microbial adhesion and aggregation.
Chronic ulcers have been shown to be caused by Helicobacter pylori and the presence of chronic ulcers is associated frequently with complications leading to gastric cancer [Cover and Blaser, 1995]. H. pylori, first isolated from a specimen of gastritis in 1983, is a gram-negative, spiral microaerophilic, fastidious organism. Generally all patients with gastric ulcer have H. pylori and eradication of the bacteria is followed by the resolution of gastritis [Cover and Blaser, 1995; Blaser, 1996]. It is estimated that H. pylori infection increases the risk of gastric cancer by approximately six times. Epidemiological studies showed that about 50% of the world population is infected with H. pylori (80% and 40% of adults in developing and developed countries, respectively) but only part of the infected population develop clinical symptoms.
The unique characteristic of H. pylori infections is persistence of the agent within the gastric mucous. The immune response against the infection is not able to eradicate the infection [Dunn et al, 1997]. Histological examination shows that the bacteria are in close association with mucus layer and underneath gastric cells [Lee et al, 1993]. The microorganisms produce a number of adhesins which mediate attachment of the bacteria to cognate receptors present in gastric mucus and epithelium [Boren and Falk, 1995; Dunn et al, 1997]. They agglutinate erythrocytes and it has been shown that the ability to adhere to gastric epithelium correlates with its ability to agglutinate red blood cells. Adhesion to gastric epithelial cells and to the mucus layer coating such surfaces is considered to be the most important factor enabling the pathogen to cause persistent infection [Boren and Falk, 1995]. It is an object of the present invention therefore to provide anti-adhesion compounds for anti-adhesion therapy which can be an alternative to the conventional antibiotic treatment.
Bacterial activity of over 500 different bacteria have been implicated in both human dental plaque and caries (cavities). Adhesion of the bacteria to each other (intraspecies) and to other bacterial species (intergeneric coaggregation) as well as to oral surfaces is one of the major factors leading to dental plaque as well as carries and periodontal diseases.
It would be useful to have additional anti-aggregation medicaments for use in oral hygiene. U.S. Pat. No. 5,362,480, columns 1-3 provides a discussion on bacterial adhesions and oral hygiene. Ofek and Doyle, 1994 provides a general discussion of bacterial adhesion incorporated herein by reference in their entirety.
Briefly, microbial accumulations on the tooth surfaces, termed dental plaque, are the causative agents of both dental caries and periodontal diseases [Slots, 1977; Socransky et al., 1982; Savit and Socransky, 1984; Dzink et al., 1985, 1988]. The adhesion of bacteria to the pellicle-coated tooth surface appears to be the first step in the formation of dental plaque [Gibbons and van Houte, 1975; van Houte, 1980]. Oral streptococci and to some extent Actinomyces sp. are the prominent early colonizers of the tooth surfaces [Nyvad and Kilian, 1990] and apparently attach to macromolecules selectively adsorbed to tooth surfaces [Gibbons et al., 1991].
Microorganisms that progressively accumulate thereafter, mostly gram negative anaerobic bacteria, in the gingival crevice area, are the late colonizers and are believed to play a central role in the initiation and progression of periodontal diseases. In this second step the bacteria co-aggregate or adhere to each other. The primary constituents of dental plaque are bacteria in a matrix composed of extracellular bacterial polymers and salivary products. The bacterial species present in dental plaque are heterogeneous and they change progressively as the clinical condition goes from normal health through gingivitis to advanced stages of periodontitis [Moore and Moore 1994].
Studies in vitro of coaggregation among oral bacteria revealed that coaggregation is essentially the result of adhesion mediated by specific interactions between complementary molecules on the surfaces of the participating bacteria [Kolenbrander et al, 1993]. Several hundreds of oral bacterial pairs were found to participate in this type of multigeneric coaggregation reactions in vitro, but only for handful of pairs the molecular mechanisms have been characterized [Ofek and Doyle, 1994]. In many cases the coaggregation involves lectin-carbohydrate interaction whereby the sugar residues on one bacterial pair interact with a lectin on the surface of the other bacterial pair.
Based on the ability of simple and complex sugars to inhibit coaggregation, a number of distinct specificities are now recognized including lactose, sialic acid, rhamnose and fucose inhibitable coaggregations. It should be noted however, that still a large number of coaggregating pairs are not inhibited by any of the carbohydrates tested and therefore they may have a distinct specificity involving surface constituents other than lectin and carbohydrate [Ofek and Doyle, 1994].
It is therefore an object of the present invention to provide compounds to inhibit interbacterial coaggregation or adhesion of oral bacteria or to reverse existing coaggregation.
There is presently anecdotal and scientific evidence that cranberry juice or some fraction thereof inhibits or reduces bacterial infections of the bladder [Avorn et al, 1994], restricted to P-fimbriated bacteria. Currently, it is believed that this action is due to interruption of the adhesion of P-fimbriated bacteria to mammalian cells.
U.S. Pat. Nos. 5,002,759 and 5,362,480 disclose anti-adhesion compositions that can be used in treating oral bacteria. However, neither of these patents disclose compositions from Vaccinium, and in particular cranberry or blueberry, and are not the composition of the present invention.
U.S. Pat. No. 5,185,153 provides a composition for use in oral compositions for the lysis and killing of oral bacteria. The '153 patent does not derive the agent from cranberry and is not the composition of the present invention.
U.S. Pat. No. 5,474,774 to Walker et al issued Dec. 12, 1995 does disclose an extract from cranberry which is enriched for an activity which inhibits bacterial adhesion to surfaces. However, the extract is not the composition of the present invention as shown in comparative Example 5 herein below. Further, the method of the '774 patent initiates the extraction from whole cranberries with multiple extraction steps. PCT/US96/03978 (WO 96/30033) published application further discloses an extract/composition. However, as shown herein below the composition of PCT/US96/03978 is not the composition of the present invention as shown in comparative Example 5 herein below.
U.S. Pat. No. 4,857,327 to Virdalm discloses a preparation isolated from the remainder of the berries after the pulp flesh has been removed. In the art of juice making the material from the Virdalm '327 is referred to as the “press cake” which is not used for making juice. Juice is derived from the “pulp flesh”. Therefore since the starting point for the Virdalm '327 product is from the remainder after discarding the pulp flesh and thereby the juice.
U.S. Pat. No. 5,683,678 to Heckert et al discloses an oral composition containing anthocyanins isolated from cranberries. However, the extract is not the composition of the present invention as shown in comparative Example 5 herein below.
It is an object of the present invention to use commercially prepared juice or concentrate from berries of the Vaccinium plant genus as the starting source of isolation with minimal extraction steps of an anti-coaggregation, adhesion fraction.